Acid phosphatase（ACP）Activity assay kit instruction manual

Micro method 100T/48S

Before formal testing, it is necessary to take 2-3 Make predictions based on samples with significant expected differencesMeasurement significance:

ACP Under acidic conditions, catalyzing the hydrolysis of phosphate monoesters is called inorganic phosphate, which is commonly found in the lysosomes of macrophages.ACP Commonly used as an auxiliary diagnosis for prostate cancer.

Measurement principle:

In acidic environments,ACP Catalytic hydrolysis of disodium p-nitrophenyl phosphate to generate 4-NitrobenPhenol, in405nm Has characteristic light absorption; through measurementfixed405nmCalculate the rate of increase in absorbanceACPActivity.

Acid phosphatase (ACP) activity assay kit esterase series

composition

Self provided instruments and supplies:

ELISA reader96 Orifice plate, desktop centrifuge, water bath, adjustable pipette, and distilled water.

The specific product parameters are subject to the parameters in the product manual received

Extraction of crude enzyme solution:

1. Organization: According to organizational quality（g）Reagent 1 volume(ml)for 1：5~10 The proportion of（Suggest weighing the agreement 0.1g Organize, join 1ml reagent1） Perform ice bath homogenization,4℃The10000gcentrifugation 10 minTake the supernatant for testing.

2. Bacteria or cells: according to the number of bacteria or cells（10⁴ One volume of reagent（ml）for 500~1000：1 The proportion of（suggestion 500 ten thousandCell addition 1ml Reagent 1), ice bath ultrasonic disruption of cells（power 300w, Ultrasound 3 Seconds, intervals 7 Second, total time 3 min）；then 10000g，4℃, centrifugation 10minTake the supernatant and place it on ice for testing.

3. Blood can be measured directly or after appropriate dilution.

determination:

1. Preheating of ELISA reader 30 minAdjust the wavelength to 405 nm.

2. in EP Add the following reagents into the tube

Mix well and absorb 200µL join 96 In the orifice plate,405 nm Measure the absorbance values of each tube.ΔA=A Measurement tube-A Take care of it.

Attention: Each measuring tube needs to have a control tube.

ACP Activity calculation:

standard curve y =7.3336x +0.0179；R² =0.9996；x For standard concentration（μmol/ml），y For absorbance valueΔA.

1. In the bloodACP Activity Calculation

Definition of active unit:30℃Catalytic production per milliliter of blood per minute 1μmol 4-Nitrophenol is defined as 1 One enzyme activity unit.

ACP 活力(μmol/min/ml)=（ΔA-0.0179）÷7.3336 ×V Anti General÷T÷V sample

= 0.4545 ×（ΔA-0.0179）

2. In organizations, bacteria, or cells ACP Activity Calculation

（1）Calculated based on protein concentration

Definition of active unit:30℃Catalytic production per minute per milligram of protein 1 μmol 4-Nitrophenol is defined as 1 One enzyme activity unit.

ACP 活力(μmol/min/mg prot)= (ΔA-0.0179)÷7.3336 ×V Anti General÷T÷(V sample×Cpr)

=0.4545 ×（ΔA-0.0179）÷Cpr

（2）Calculated based on sample quality

Definition of active unit:30℃Catalytic production per minute per gram of tissue 1 M mol 4-Nitrophenol is defined as 1 One enzyme activity unit.

ACP 活力 (μmol/min/g fresh weight)=(ΔA-0.0179)÷7.3336 ×V Anti General÷T÷(W×V sample÷V Sample General)

= 0.4545 ×（ΔA-0.0179）÷W

（3）Calculated based on the number of bacteria or cells

Definition of active unit:30℃Every middle 10⁴ Bacteria or cells catalyze production every minute 1 M mol 4-Nitrophenol is defined as 1 One enzyme activity unit.

ACP 活力(μmol/min/10⁴ cell)=(ΔA-0.0179)÷7.3336 ×V Anti General÷T÷(Number of cells)×V sample÷V Sample General）

=0.4545 ×（ΔA-0.0179）÷cell count

VCounter total: total volume of the reaction system（ml），1ml； W : Sample quality,g； V Sample: Add sample volume to the reaction system（ml），

0.01ml；V Sample total: volume of extraction solution,1ml；TResponse time（min），30 min；500: Total number of cells or bacteria,500 Wan.

Notes:

ACP Unstable, especially in 37℃andpH greater than 7 Under the condition of rapid loss of vitality, acid phosphatase samples generally need to be prepared on the same day;Add to every milliliter of serum sample 10mgSodium hydrogen citrate or 5mgSodium bisulfate, to makepHdrop to 6.5 Below, or 5mlbloodQing joined 30%Acetic acid solution 2～3 Drop, place it 4℃Can be saved 1 week

Acid phosphatase (ACP) activity assay kit esterase series